THE THERMODYNAMICS OF ASSOCIATION AND UNFOLDING OF THE 205-316-C-TERMINAL FRAGMENT OF THERMOLYSIN

The 205-316 C-terminal fragment of thermolysin has been studied by dif ferential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimenta l conditions. The effect of sample concentration at the different pH v alues on the calorimetric traces is consistent with a monomer-dimer eq uilibrium of the folded fragment, which undergoes thermal unfolding in to individual fragments. Equilibrium sedimentation experiments at 10 d egrees C and different pH values confirm the presence of the associati on equilibrium and provide the value of the dimerization constants. Th e global analysis of the calorimetric, heat capacity curves has been c arried out by a multidimensional fitting to the model N-2 reversible a rrow 2N reversible arrow 2U. The analysis leads to a complete thermody namic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a part ially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information curre ntly available and compared with the energetics of unfolding of the sh orter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lar a, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases th e relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment.